Two isoforms of thioredoxin reductase (TR) from HeLa cells, differing in their affinity to heparin and rat liver TR antibodies, were studied by spectrophotometry, atomic absorption, and electrospray ionization mass spectrometry. The enzyme, which has a selenocysteine followed by glycine at its C-terminal, plays an important role in cell metabolism regulation and proliferation. High heparin affinity isoform (HHAI) proved to have half the amount of selenium per mol of protein and about one third of the specific catalytic activity of the low heparin affinity isoform (LHAI). LHAI is always more oxidized than HHAI under similar conditions. SDS PAGE and immunoblot assay with antibodies elicited to synthetic peptide, corresponding to V433-A452 of the predicted protein sequence of human placenta TR, did not show a significant difference between the two isoforms. However, extended incubation with rat liver TR polyclonal antibodies revealed, that HHAI consists of two closely migrating bands, the lower of which does not contain selenium. The isoelectric point value of this isoform was 5.85, which is different from 5.2-5.4 for LHAI. Molecular mass of intact LHAI, calculated from electrospray ionization mass spectra, is 54800 Da, which is 419 Da higher than that predicted for mature enzyme. This implies its possible modification. Alkylation of the intact isoform at pH 6.5 revealed that both Cys 495 and Secys 496 are ionized in NADPH reduced enzyme and probably play an important role in catalytic activity of TR.